Quantitative measurements of the size and concentration of macromolecules, such as proteins and DNA, is useful for many biological assays as well as studies of colloidal and macromolecular solutions. Traditionally, this has been accomplished through ultracentrifugation, chromatography, and especially, gel electrophoresis. See, Alberts et al. (1994) Molecular Biology of the Cell, Garland Publishing, Inc., NY.
Coulter counters typically consist of two fluid-filled reservoirs of particle-laden solution separated by a membrane and connected by a small aperture or pore in that membrane. Particles in the solution are driven through the pore and in doing so, displace conducting fluid and raise the electrical resistance of the pore. By monitoring the changes in electrical current through the pore as individual particles pass from one reservoir to the other, Coulter counters are able to measure the sizes of particles passing through the pore.
While this method has long been used to characterize solutions of micron- (or greater) size cells, its relative simplicity has led to efforts to employ it to detect sub-micron particles, including viruses. See, Kubitschak (1968) Nature 182:234; Gregg et al. (1965) Biophys. J. 5:393; DeBlois et al. (1970) Rev. Sci. Instrum. 41:909; and DeBlois et al. (1977) J. Colloid Interface Sci. 61:323.
Coulter conduits typically having diameters ranging between approximately 0.015 mm and 0.200 mm, with conduit length-to-diameter ratios L/D between 0.75 and 1.2, have proven useful for a great variety of particles. Resolutions down to particle sizes of 0.6 μm have been reported.
Sun and Crooks have proposed a method for fabricating single pores in a gold membrane. However, use of the gold membrane results in problematic charging effects, which reduce the time resolution of measurements. See, Sun et al. (1999) Langmuir 15:738. Moreover, their device utilizes a pore that runs from one side to the other side of the substrate.
Koch et al. (1999) J. Micromech. Microeng. 9:159 describes a device having a pore of ˜5 micron diameter. The device is capped with a glass sheet which must be adhered to the substrate via high-temperature electrical bonding procedures. Moreover, the fluid access in the Koch device is from the backside of the chip.
Genetically-modified transmembrane protein pores, suspended in lipid bilayers and molecular-scaled holes in silicon nitride have also been used as engineered nanopores. However, these strategies suffer from difficulties in creating an effective and stable pore or array of pores. See, Howorka et al. (201) Nature Biotechnology 19:636.
Miniaturization of a Coulter counter would allow for other applications such as the characterization of biological cells, e.g., red blood cells, colloids, or even biological molecules. It is desirable that simple apparatus for sensing and characterizing particles be provided which offers increased sensitivity to particle characteristics.